Narrow-distribution PLA (polylactic acid) microspheres as well as preparation method and application thereof

Abstract

The embodiment of the invention provides narrow-distribution PLA (polylactic acid) microspheres as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving a PLA material in an organic solvent to obtain a dispersed phase; based on the total volume of the dispersion phase, the concentration of the PLA material in the dispersion phase is 60-300 mg / mL; dissolving polyvinyl alcohol in hot water to obtain a continuous phase; the mass concentration of the polyvinyl alcohol in the continuous phase is 1.5-5% by taking the total weight of the hot water as 100%; enabling the dispersed phase and the continuous phase to form a stable primary emulsion by adopting a porous membrane tube through a membrane emulsification method; stirring the primary emulsion under an ice bath condition at the temperature of 10 + / -2 DEG C to obtain a cured emulsion; and sequentially centrifuging, screening, collecting and freeze-drying the cured emulsion to obtain the narrow-distribution PLA microspheres. The PLA microspheres prepared by the preparation method provided by the invention are smooth and regular in surface, free of cracks, not easy to adhere, narrow in particle size distribution and high in yield.

Description

Technical Field

[0001] The invention relates to a narrow distribution PLA microsphere and a preparation method and application thereof, belonging to the technical field of biomedical engineering. Background Art

[0002] As humans age or are affected by certain diseases, the muscles and collagen tissues in the human body will experience varying degrees of functional degradation, causing problems such as skin atrophy and increased wrinkles. Therefore, people have invented a variety of fillers, such as hyaluronic acid, bovine collagen, etc., but the filling effect of these substances as fillers is short-lived and requires frequent injections to maintain their filling effect. In order to achieve long-term filling effects, people have tried to use microspheres made of slow-degrading or non-degradable materials, such as polyvinyl alcohol, polymethyl methacrylate, etc. as fillers. Although the filling effect of fillers made of such materials lasts significantly longer, these materials will also be corroded or degraded in the body over time, and release harmful substances, causing side effects that endanger human health.

[0003] Polylactic acid (PLA) is a high molecular polymer with good biocompatibility and biodegradability, which has been widely used in the fields of tissue engineering, drug controlled release and plastic surgery. At present, the commonly used methods for making PLA microspheres mainly include mechanical stirring emulsification method and spray drying method, but the PLA microspheres prepared by these methods have rough surfaces, cracks, wide particle size distribution, easy adhesion and low microsphere yield.

[0004] In the process of realizing the concept of the present invention, the inventors found that there are at least the following problems in the related art: the existing commonly used methods for producing PLA microspheres mainly include mechanical stirring emulsification method and spray drying method, but the PLA microspheres prepared by these methods have a rough surface, cracks, a wide particle size distribution, easy adhesion and low microsphere yield.

[0005] Therefore, it is necessary to provide a new type of narrow distribution PLA microspheres and their preparation method and application, which has become a technical problem that needs to be solved urgently in this field. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a narrow distribution PLA microsphere and a preparation method and application thereof, which solves the technical problems that the PLA microspheres prepared by the existing conventional preparation method have a rough surface, cracks, a wide particle size distribution, easy adhesion and a low microsphere yield.

[0007] In order to solve the above technical problems, a specific embodiment of the present invention provides a method for preparing narrow distribution PLA microspheres, wherein the preparation method comprises:

[0008] Step (1): dissolving a PLA material in an organic solvent to obtain a dispersed phase; based on the total volume of the dispersed phase, the concentration of the PLA material in the dispersed phase is 60 to 300 mg / mL;

[0009] Step (2): dissolving polyvinyl alcohol in hot water to obtain a continuous phase; based on the total weight of the hot water as 100%, the mass concentration of the polyvinyl alcohol in the continuous phase is 1.5-5%;

[0010] Step (3): using a porous membrane tube to form a stable primary emulsion from the dispersed phase and the continuous phase through a membrane emulsification method;

[0011] Step (4): stirring the primary emulsion in an ice bath at a temperature of 10±2° C. to remove the organic solvent and obtain a solidified emulsion;

[0012] Step (5): the solidified emulsion is centrifuged, sieved and collected, and freeze-dried in sequence to obtain the narrow distribution PLA microspheres.

[0013] In one embodiment of the present invention, in step (1), the concentration of the PLA material in the dispersed phase is 100-300 mg / mL, preferably 100 mg / mL, based on the total volume of the dispersed phase.

[0014] In one embodiment of the present invention, in step (1), the weight average molecular weight of the PLA material is 15 to 250 kDa.

[0015] In one embodiment of the present invention, in step (1), the PLA material includes one or a combination of left-handed polylactic acid (PLLA), right-handed polylactic acid (PDLA), racemic polylactic acid (PDLLA) and meso-polylactic acid (meso-PLA).

[0016] In one embodiment of the present invention, in step (1), the organic solvent includes one or a combination of dichloromethane, chloroform, methyl formate, ethyl acetate, methyl acetate, acetone, dichloroethane and pentane, etc.; preferably dichloromethane.

[0017] In one embodiment of the present invention, in step (2), the hot water includes drinking water, purified water or water for injection, etc., preferably purified water.

[0018] The present invention does not make specific requirements on the temperature of the hot water used in step (2) of the preparation method described above, and can be reasonably selected and adjusted as needed, as long as the hot water used can fully dissolve the polyvinyl alcohol and obtain a continuous phase that meets the requirements. For example, in some embodiments of the present invention, the temperature of the hot water can be 80°C.

[0019] In one embodiment of the present invention, in step (2), based on the total weight of the continuous phase being 100%, the mass percentage of polyvinyl alcohol in the continuous phase is 1.5-3%.

[0020] In one embodiment of the present invention, in step (3), the volume ratio of the dispersed phase to the continuous phase is 1:2 to 200, preferably 1:10.

[0021] In one embodiment of the present invention, in step (3), the pore size of the porous membrane tube is 0.1-20 μm, preferably 4-10 μm, and more preferably 7 μm.

[0022] In one embodiment of the present invention, in step (3), the porous membrane tube is a porous glass membrane tube.

[0023] In one embodiment of the present invention, step (4) includes the following specific steps:

[0024] The three-necked flask is fixed in a fume hood, an overhead stirrer is set up, a polytetrafluoroethylene stirring paddle is inserted into the stirrer, and the polytetrafluoroethylene stirring paddle is extended into the primary emulsion through the middle opening of the three-necked flask, and a side opening of the three-necked flask is connected to a circulating water vacuum pump;

[0025] The primary emulsion was poured into the three-necked flask through the other side opening of the three-necked flask and the side opening was plugged with a glass piston. The primary emulsion was stirred in an ice bath at 10±2° C. to remove the organic solvent and obtain a solidified emulsion.

[0026] In one embodiment of the present invention, in step (4), the polytetrafluoroethylene stirring paddle extends into the primary emulsion at a position of 1 / 4-3 / 4 of the height of the primary emulsion, preferably 1 / 2;

[0027] And / or the stirring speed is 120-180 r / min, preferably 150 r / min, and the stirring time is 8-16 h, preferably 12 h.

[0028] The present invention does not make specific requirements on the specific operations of centrifugation, sieving collection and freeze-drying in step (5) of the preparation method described above, as well as the process parameters such as temperature, time and rotation speed used, and can be reasonably selected and adjusted as needed, as long as the corresponding purpose of the operation can be achieved.

[0029] A specific embodiment of the present invention further provides a narrow distribution PLA microsphere, wherein the narrow distribution PLA microsphere is prepared by the above-mentioned preparation method of narrow distribution PLA microsphere, and has a particle size range of 10 to 30 μm, and a diameter distance of no more than 0.6, preferably no more than 0.560.

[0030] The specific embodiments of the present invention also provide the use of the narrow distribution PLA microspheres as cosmetic plastic surgery fillers, as drug carriers for preparing sustained-release drugs, or as raw materials for preparing tissue engineering scaffolds.

[0031] Compared with the prior art, the technical effects achieved by the technical solution of the present invention include at least:

[0032] The preparation method of narrow distribution PLA microspheres provided by the present invention first uses a porous membrane tube to form a stable primary emulsion with a dispersed phase and a continuous phase through a membrane emulsification method; then, the primary emulsion is stirred under an ice bath condition of 10±2°C to remove the organic solvent to obtain a solidified emulsion; finally, the solidified emulsion is centrifuged, sieved, collected and freeze-dried in sequence to obtain the narrow distribution PLA microspheres. Among them, the low-temperature ice bath at a temperature of 10±2°C can slowly release organic solvents such as dichloromethane, thereby avoiding cracks or wrinkles on the surface of the microspheres, and stirring the primary emulsion can reduce collisions between emulsion droplets, thereby making it less likely to stick together.

[0033] In summary, the PLA microspheres prepared by the preparation method provided by the present invention have a smooth and regular surface, no cracks, are not easy to stick together, have a narrow particle size distribution, and have a high yield of PLA microspheres.

[0034] It should be understood that the above general description and the following detailed description are merely exemplary and illustrative and are not intended to limit the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings below are part of the specification of the present invention, and illustrate exemplary embodiments of the present invention. Together with the description of the specification, the accompanying drawings are used to explain the principles of the present invention.

[0036] Figure 1 This is an electron microscope image of the PLA microspheres provided in Example 1 of the present invention.

[0037] Figure 2 This is an electron microscope image of the PLA microspheres provided in Example 2 of the present invention.

[0038] Figure 3 This is an electron microscope image of the PLA microspheres provided in Example 3 of the present invention.

[0039] Figure 4 This is an electron microscope image of the PLA microspheres provided for Comparative Example 1.

[0040] Figure 5 This is an electron microscope image of the PLA microspheres provided for Comparative Example 2.

[0041] Figure 6 This is an electron microscope image of the PLA microspheres provided for Comparative Example 3. DETAILED DESCRIPTION

[0042] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be clearly explained with the accompanying drawings and detailed descriptions below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0043] The exemplary embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention. In addition, elements / components with the same or similar reference numerals used in the drawings and embodiments are used to represent the same or similar parts.

[0044] The terms “first”, “second”, etc. used in this document do not particularly refer to an order or sequence, nor are they used to limit the present invention. They are only used to distinguish elements or operations described with the same technical terms.

[0045] The directional terms used in this document, such as up, down, left, right, front or back, etc., are only used to refer to the directions of the drawings. Therefore, the directional terms used are used to illustrate and not to limit the present invention.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] As used herein, "and / or" includes any or all combinations of the items described.

[0048] As used herein, “plurality” includes “two” and “more than two”; as used herein, “plurality groups” includes “two groups” and “more than two groups”.

[0049] The terms "substantially" and "approximately" used herein are used to modify any quantity or error that may vary slightly, but these slight changes or errors do not change their essence. Generally speaking, the range of slight changes or errors modified by such terms may be 20% in some embodiments, 10% in some embodiments, 5% in some embodiments, or other values. Those skilled in the art should understand that the aforementioned values ​​can be adjusted according to actual needs and are not limited thereto.

[0050] All terms (including technical and scientific terms) used herein have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein should be interpreted as having a meaning consistent with the context of this specification, and should not be interpreted in an idealized or overly rigid manner.

[0051] In the case of using expressions such as "at least one of A, B, and C, etc.", it should generally be interpreted in accordance with the meaning of the expression generally understood by those skilled in the art (for example, "a system having at least one of A, B, and C" should include but not be limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.). In the case of using expressions such as "at least one of A, B, or C, etc.", it should generally be interpreted in accordance with the meaning of the expression generally understood by those skilled in the art (for example, "a system having at least one of A, B, or C" should include but not be limited to a system having A alone, B alone, C alone, A and B, A and C, B and C, and / or A, B, C, etc.). Those skilled in the art should also understand that any transitional conjunctions and / or phrases that substantially indicate two or more optional items, whether in the specification, claims, or drawings, should be understood to give the possibility of including one of these items, either of these items, or both of these items. For example, the phrase "A or B" should be understood to include the possibilities of "A" or "B", or "A and B".

[0052] "Scope" disclosed in the present invention is given in the form of lower limit and upper limit. It can be one or more lower limits, and one or more upper limits respectively. A given range is defined by selecting a lower limit and an upper limit. The selected lower limit and upper limit define the boundaries of a particular range. All ranges defined in this way are combinable, i.e. any lower limit can be combined with any upper limit to form a range. For example, for a specific parameter, a range of 60-120 and 80-110 is listed, and it is understood that a range of 60-110 and 80-120 is also expected. In addition, if the minimum range values ​​listed are 1 and 2, and the maximum range values ​​listed are 3, 4 and 5, then the following ranges can all be expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5.

[0053] In the present invention, unless otherwise specified, the numerical range "ab" represents an abbreviation of any real number combination between a and b, where a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed in the present invention, and "0-5" is just an abbreviation of these numerical combinations.

[0054] In the present invention, unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0055] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0056] Example 1

[0057] This embodiment provides a narrow distribution PLA microsphere, which is prepared by a preparation method comprising the following specific steps:

[0058] Step (1): using dichloromethane as solvent, a certain amount of PLLA (poly(lactic acid) with a weight average molecular weight of 15-25 kDa) is dissolved in dichloromethane to obtain a dispersed phase; based on the total volume of the dispersed phase, the concentration of PLLA in the dispersed phase is 100 mg / mL.

[0059] Step (2): 16 g of polyvinyl alcohol (PVA) was dissolved in 800 mL of purified water at 80° C. to prepare a continuous phase with a PVA mass concentration of 2%.

[0060] Step (3): placing a porous membrane tube (porous glass membrane tube, the pore size of the membrane is 7 μm) in the continuous phase, with the lower end of the porous membrane tube 2 cm away from the magnetic stirrer in the continuous phase, and the center of the porous membrane tube is aligned with the edge of the magnetic stirrer, and the rotation speed of the magnetic stirrer is 800 r / min;

[0061] Add 30 mL of dispersed phase, introduce nitrogen, wait for the pressure to rise slowly, control the working pressure at 0.007±0.001 MPa, allow the dispersed phase to pass through (press through) the membrane pores at a constant flow rate, form dispersed phase droplets in the continuous phase, use a gear pump to pump the continuous phase to the membrane tube at a certain flow rate, take away the dispersed phase droplets, and form a stable primary emulsion.

[0062] Step (4): fix the three-necked flask in a fume hood, set up an overhead stirrer, insert a polytetrafluoroethylene stirring paddle into the stirrer, and extend the polytetrafluoroethylene stirring paddle into the primary emulsion through the middle opening of the three-necked flask, and the position where the polytetrafluoroethylene stirring paddle extends into the primary emulsion is 1 / 2 of the height of the primary emulsion, and connect one side end opening of the three-necked flask to a circulating water vacuum pump;

[0063] The primary emulsion was poured into the three-necked flask through the other side opening of the three-necked flask, and the side opening was plugged with a 24 / 29 glass piston. The primary emulsion was stirred at an ice bath temperature of 10±2° C. at a stirring speed of 150 r / min for 12 h to remove (volatilize) the organic solvent, thereby obtaining a solidified emulsion;

[0064] Step (5): Transfer the solidified emulsion to a centrifuge tube, set the speed to 1500 rpm, centrifuge for 3 minutes, and pour out the supernatant; then add purified water to the centrifuge tube to 2 / 3 of the volume of the centrifuge tube, set the speed to 1500 rpm, continue centrifugation for 3 minutes, pour out the supernatant, and observe carefully. When the white microsphere suspension is 2 cm away from the tube mouth, stop pouring. Repeat this three times.

[0065] The microspheres were collected by sieving and finally freeze-dried to obtain PLA microspheres.

[0066] Example 2

[0067] This embodiment provides a narrow distribution PLA microsphere, which is prepared by a preparation method comprising the following specific steps:

[0068] Step (1): using dichloromethane as solvent, a certain amount of PLLA (poly(lactic acid) with a weight average molecular weight of 75-125 kDa) is dissolved in dichloromethane to obtain a dispersed phase; based on the total volume of the dispersed phase, the concentration of PLLA in the dispersed phase is 100 mg / mL.

[0069] Step (2): 16 g of polyvinyl alcohol (PVA) was dissolved in 800 mL of purified water at 80° C. to prepare a continuous phase with a PVA mass concentration of 2%.

[0070] Step (3): placing a porous membrane tube (porous glass membrane tube, the pore size of the membrane is 7 μm) in the continuous phase, with the lower end of the porous membrane tube 2 cm away from the magnetic stirrer in the continuous phase, and the center of the porous membrane tube is aligned with the edge of the magnetic stirrer, and the rotation speed of the magnetic stirrer is 800 r / min;

[0071] Add 30 mL of dispersed phase, introduce nitrogen, wait for the pressure to rise slowly, control the working pressure at 0.007±0.001 MPa, allow the dispersed phase to pass through (press through) the membrane pores at a constant flow rate, form dispersed phase droplets in the continuous phase, use a gear pump to pump the continuous phase to the membrane tube at a certain flow rate, take away the dispersed phase droplets, and form a stable primary emulsion.

[0072] Step (4): fix the three-necked flask in a fume hood, set up an overhead stirrer, insert a polytetrafluoroethylene stirring paddle into the stirrer, and extend the polytetrafluoroethylene stirring paddle into the primary emulsion through the middle opening of the three-necked flask, and the position where the polytetrafluoroethylene stirring paddle extends into the primary emulsion is 1 / 2 of the height of the primary emulsion, and connect one side end opening of the three-necked flask to a circulating water vacuum pump;

[0073] The primary emulsion was poured into the three-necked flask through the other side opening of the three-necked flask, and the side opening was plugged with a 24 / 29 glass piston. The primary emulsion was stirred for 12 hours at a stirring speed of 150 r / min in an ice bath at a temperature of 10±2°C to remove (volatilize) the organic solvent to obtain a solidified emulsion.

[0074] Step (5): Transfer the solidified emulsion to a centrifuge tube, set the speed to 1500 rpm, centrifuge for 3 minutes, and pour out the supernatant; then add purified water to the centrifuge tube to 2 / 3 of the volume of the centrifuge tube, set the speed to 1500 rpm, continue centrifugation for 3 minutes, pour out the supernatant, and observe carefully. When the white microsphere suspension is 2 cm away from the tube mouth, stop pouring. Repeat this three times.

[0075] The microspheres were collected by sieving and finally freeze-dried to obtain PLA microspheres.

[0076] Example 3

[0077] This embodiment provides a narrow distribution PLA microsphere, which is prepared by a preparation method comprising the following specific steps:

[0078] Step (1): using dichloromethane as solvent, a certain amount of PLLA (poly(lactic acid) with a weight average molecular weight of 150-250 kDa) is dissolved in dichloromethane to obtain a dispersed phase; based on the total volume of the dispersed phase, the concentration of PLLA in the dispersed phase is 100 mg / mL.

[0079] Step (2): 16 g of polyvinyl alcohol (PVA) was dissolved in 800 mL of purified water at 80° C. to prepare a continuous phase with a PVA mass concentration of 2%.

[0080] Step (3): placing a porous membrane tube (porous glass membrane tube, the pore size of the membrane is 7 μm) in the continuous phase, with the lower end of the porous membrane tube 2 cm away from the magnetic stirrer in the continuous phase, and the center of the porous membrane tube is aligned with the edge of the magnetic stirrer, and the rotation speed of the magnetic stirrer is 800 r / min;

[0081] Add 30 mL of dispersed phase, introduce nitrogen, wait for the pressure to rise slowly, control the working pressure at 0.007±0.001 MPa, allow the dispersed phase to pass through (press through) the membrane pores at a constant flow rate, form dispersed phase droplets in the continuous phase, use a gear pump to pump the continuous phase to the membrane tube at a certain flow rate, take away the dispersed phase droplets, and form a stable primary emulsion.

[0082] Step (4): fix the three-necked flask in a fume hood, set up an overhead stirrer, insert a polytetrafluoroethylene stirring paddle into the stirrer, and extend the polytetrafluoroethylene stirring paddle into the primary emulsion through the middle opening of the three-necked flask, and the position where the polytetrafluoroethylene stirring paddle extends into the primary emulsion is 1 / 2 of the height of the primary emulsion, and connect one side end opening of the three-necked flask to a circulating water vacuum pump;

[0083] The primary emulsion was poured into the three-necked flask through the other side opening of the three-necked flask, and the side opening was plugged with a 24 / 29 glass piston. The primary emulsion was stirred for 12 hours at a stirring speed of 150 r / min in an ice bath at a temperature of 10±2°C to remove (volatilize) the organic solvent to obtain a solidified emulsion.

[0084] Step (5): Transfer the solidified emulsion to a centrifuge tube, set the speed to 1500 rpm, centrifuge for 3 minutes, and pour out the supernatant; then add purified water to the centrifuge tube to 2 / 3 of the volume of the centrifuge tube, set the speed to 1500 rpm, continue centrifugation for 3 minutes, pour out the supernatant, and observe carefully. When the white microsphere suspension is 2 cm away from the tube mouth, stop pouring. Repeat this three times.

[0085] The microspheres were collected by sieving and finally freeze-dried to obtain PLA microspheres.

[0086] Comparative Example 1

[0087] This comparative example provides a narrow distribution PLA microsphere, which is prepared by a preparation method comprising the following specific steps:

[0088] Step (1): using dichloromethane as solvent, a certain amount of PLLA (poly(lactic acid) with a weight average molecular weight of 15-25 kDa) is dissolved in dichloromethane to obtain a dispersed phase; based on the total volume of the dispersed phase, the concentration of PLLA in the dispersed phase is 100 mg / mL.

[0089] Step (2): 8 g of polyvinyl alcohol (PVA) was dissolved in 800 mL of purified water at 80° C. to prepare a continuous phase with a PVA mass concentration of 1%.

[0090] Step (3): placing a porous membrane tube (porous glass membrane tube, the pore size of the membrane is 7 μm) in the continuous phase, with the lower end of the porous membrane tube 2 cm away from the magnetic stirrer in the continuous phase, and the center of the porous membrane tube is aligned with the edge of the magnetic stirrer, and the rotation speed of the magnetic stirrer is 800 r / min;

[0091] Add 30 mL of dispersed phase, introduce nitrogen, wait for the pressure to rise slowly, control the working pressure at 0.007±0.001 MPa, allow the dispersed phase to pass through (press through) the membrane pores at a constant flow rate, form dispersed phase droplets in the continuous phase, use a gear pump to pump the continuous phase to the membrane tube at a certain flow rate, take away the dispersed phase droplets, and form a stable primary emulsion.

[0092] Step (4): fix the three-necked flask in a fume hood, set up an overhead stirrer, insert a polytetrafluoroethylene stirring paddle into the stirrer, and extend the polytetrafluoroethylene stirring paddle into the primary emulsion through the middle opening of the three-necked flask, and the position where the polytetrafluoroethylene stirring paddle extends into the primary emulsion is 1 / 2 of the height of the primary emulsion, and connect one side end opening of the three-necked flask to a circulating water vacuum pump;

[0093] The primary emulsion was poured into the three-necked flask through the other side opening of the three-necked flask, and the side opening was plugged with a 24 / 29 glass piston. The primary emulsion was stirred for 12 hours at a stirring speed of 150 r / min in an ice bath at a temperature of 10±2°C to remove (volatilize) the organic solvent to obtain a solidified emulsion.

[0094] Step (5): Transfer the solidified emulsion to a centrifuge tube, set the speed to 1500 rpm, centrifuge for 3 minutes, and pour out the supernatant; then add purified water to the centrifuge tube to 2 / 3 of the volume of the centrifuge tube, set the speed to 1500 rpm, continue centrifugation for 3 minutes, pour out the supernatant, and observe carefully. When the white microsphere suspension is 2 cm away from the tube mouth, stop pouring. Repeat this three times.

[0095] The microspheres were collected by sieving and finally freeze-dried to obtain PLA microspheres.

[0096] Comparative Example 2

[0097] This comparative example provides a narrow distribution PLA microsphere, which is prepared by a preparation method comprising the following specific steps:

[0098] Step (1): using dichloromethane as solvent, dissolving a certain amount of PLLA (poly(lactic acid) with a weight average molecular weight of 15-25 kDa) in dichloromethane to obtain a dispersed phase; based on the total volume of the dispersed phase, the concentration of PLLA in the dispersed phase is 50 mg / mL.

[0099] Step (2): 8 g of polyvinyl alcohol (PVA) was dissolved in 800 mL of purified water at 80° C. to prepare a continuous phase with a PVA mass concentration of 1%.

[0100] Step (3): placing a porous membrane tube (porous glass membrane tube, the pore size of the membrane is 7 μm) in the continuous phase, with the lower end of the porous membrane tube 2 cm away from the magnetic stirrer in the continuous phase, and the center of the porous membrane tube is aligned with the edge of the magnetic stirrer, and the rotation speed of the magnetic stirrer is 800 r / min;

[0101] Add 30 mL of dispersed phase, introduce nitrogen, wait for the pressure to rise slowly, control the working pressure at 0.007±0.001 MPa, allow the dispersed phase to pass through (press through) the membrane pores at a constant flow rate, form dispersed phase droplets in the continuous phase, use a gear pump to pump the continuous phase to the membrane tube at a certain flow rate, take away the dispersed phase droplets, and form a stable primary emulsion.

[0102] Step (4): fix the three-necked flask in a fume hood, set up an overhead stirrer, insert a polytetrafluoroethylene stirring paddle into the stirrer, and extend the polytetrafluoroethylene stirring paddle into the primary emulsion through the middle opening of the three-necked flask, and the position where the polytetrafluoroethylene stirring paddle extends into the primary emulsion is 1 / 2 of the height of the primary emulsion, and connect one side end opening of the three-necked flask to a circulating water vacuum pump;

[0103] The primary emulsion was poured into the three-necked flask through the other side opening of the three-necked flask, and the side opening was plugged with a 24 / 29 glass piston. The primary emulsion was stirred for 12 hours at a stirring speed of 150 r / min in an ice bath at a temperature of 10±2°C to remove (volatilize) the organic solvent to obtain a solidified emulsion.

[0104] Step (5): Transfer the solidified emulsion to a centrifuge tube, set the speed to 1500 rpm, centrifuge for 3 minutes, and pour out the supernatant; then add purified water to the centrifuge tube to 2 / 3 of the volume of the centrifuge tube, set the speed to 1500 rpm, continue centrifugation for 3 minutes, pour out the supernatant, and observe carefully. When the white microsphere suspension is 2 cm away from the tube mouth, stop pouring. Repeat this three times.

[0105] The microspheres were collected by sieving and finally freeze-dried to obtain PLA microspheres.

[0106] Comparative Example 3

[0107] This comparative example provides a PLA microsphere, which is prepared by a preparation method comprising the following specific steps:

[0108] Step (1): using dichloromethane as solvent, a certain amount of PLLA (poly(lactic acid) with a weight average molecular weight of 15-25 kDa) is dissolved in dichloromethane to obtain a dispersed phase; based on the total volume of the dispersed phase, the concentration of PLLA in the dispersed phase is 100 mg / mL.

[0109] Step (2): 8 g of polyvinyl alcohol (PVA) was dissolved in 800 mL of purified water at 80° C. to prepare a continuous phase with a PVA mass concentration of 1%.

[0110] Step (3): placing a porous membrane tube (porous glass membrane tube, the pore size of the membrane is 7 μm) in the continuous phase, with the lower end of the porous membrane tube 2 cm away from the magnetic stirrer in the continuous phase, and the center of the porous membrane tube is aligned with the edge of the magnetic stirrer, and the rotation speed of the magnetic stirrer is 800 r / min;

[0111] Add 30 mL of dispersed phase, introduce nitrogen, wait for the pressure to rise slowly, control the working pressure at 0.007±0.001 MPa, allow the dispersed phase to pass through (press through) the membrane pores at a constant flow rate, form dispersed phase droplets in the continuous phase, use a gear pump to pump the continuous phase to the membrane tube at a certain flow rate, take away the dispersed phase droplets, and form a stable primary emulsion.

[0112] Step (4): fix the three-necked flask in a fume hood, set up an overhead stirrer, insert a polytetrafluoroethylene stirring paddle into the stirrer, and extend the polytetrafluoroethylene stirring paddle into the primary emulsion through the middle opening of the three-necked flask, and the position where the polytetrafluoroethylene stirring paddle extends into the primary emulsion is 1 / 2 of the height of the primary emulsion, and connect one side end opening of the three-necked flask to a circulating water vacuum pump;

[0113] The primary emulsion was poured into the three-necked flask through the other side opening of the three-necked flask, and the side opening was plugged with a 24 / 29 glass piston. The primary emulsion was stirred for 12 hours at a stirring speed of 150 r / min in an ice bath at a temperature of 30±2°C to remove (volatilize) the organic solvent to obtain a solidified emulsion.

[0114] Step (5): Transfer the solidified emulsion to a centrifuge tube, set the speed to 1500 rpm, centrifuge for 3 minutes, and pour out the supernatant; then add purified water to the centrifuge tube to 2 / 3 of the volume of the centrifuge tube, set the speed to 1500 rpm, continue centrifugation for 3 minutes, pour out the supernatant, and observe carefully. When the white microsphere suspension is 2 cm away from the tube mouth, stop pouring. Repeat this three times.

[0115] The microspheres were collected by sieving and finally freeze-dried to obtain PLA microspheres.

[0116] Test Example 1

[0117] In this test example, the PLA microspheres provided by Examples 1 to 3 of the present invention and Comparative Examples 1 to 3 were dispersed in water, and then observed using an electron microscope. The electron microscope images obtained were as follows: Figure 1-Figure 6 As shown. Figure 1-Figure 3 It can be seen that the surface of the PLA microspheres provided in Examples 1 to 3 of the present invention is smooth and regular, without cracks or adhesion. Figure 4 It can be seen that the surface of the PLA microspheres provided in Comparative Example 1 collapsed and broke. Figure 5 It can be seen that cracks and wrinkles exist on the surface of the PLA microspheres provided in Comparative Example 2. Figure 6 It can be seen that cracks and wrinkles also exist on the surface of the PLA microspheres provided in Comparative Example 3. This indicates that when preparing PLA microspheres, the concentration of the PLA material in the dispersed phase is not within the range claimed in the present application, the mass concentration of the polyvinyl alcohol in the continuous phase is not within the range claimed in the present application, and / or the organic solvent is not removed under ice bath conditions, PLA microspheres that meet the requirements cannot be obtained, that is, PLA microspheres with smooth and regular surfaces, no cracks, and not easy to stick cannot be obtained.

[0118] Test Example 2

[0119] In this test example, a particle size analyzer was used to detect the particle sizes of the PLA microspheres provided in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3, respectively. The results showed that the particle sizes of the PLA microspheres provided in Examples 1 to 3 of the present invention and Comparative Examples 1 to 3 were all in the range of 10-30 μm, but their particle size distribution results and yields were different, as shown in Table 1 below.

[0120] Table 1 PLA microsphere particle size distribution and yield of Example 1-Example 3 and Comparative Example 1-Comparative Example 3

[0121]

[0122] It can be seen from Table 1 above that the yields of PLA microspheres provided by Examples 1 to 3 of the present invention are all above 90%, which are significantly higher than those of Comparative Examples 1 to 3.

[0123] In addition, it can be seen from Table 1 above that the diameters of the PLA microspheres provided in Examples 1 to 3 of the present invention are all below 0.560, and the consistency is all below 0.170, indicating that the particle size distribution of these PLA microspheres is narrower and the uniformity is better.

[0124] Compared with Comparative Examples 1 to 3, the diameter distance and consistency data of the PLA microspheres provided in Example 1 of the present invention are lower, indicating that the particle size distribution of the PLA microspheres is narrower and the uniformity is better, which further proves that when preparing PLA microspheres, the concentration of the PLA material in the dispersed phase is not within the scope claimed for protection in this application, the mass concentration of polyvinyl alcohol in the continuous phase is not within the scope claimed for protection in this application, and / or the organic solvent is not removed under ice bath conditions, and the obtained PLA microspheres have a wide particle size distribution and poor uniformity.

[0125] In summary, the embodiment of the present invention first uses a porous membrane tube to form a stable primary emulsion with a dispersed phase and a continuous phase through a membrane emulsification method; then, the primary emulsion is stirred under an ice bath condition at a temperature of 10±2°C to remove the organic solvent to obtain a solidified emulsion; finally, the solidified emulsion is centrifuged, sieved, collected and freeze-dried in sequence to obtain the narrow distribution PLA microspheres. Among them, a low-temperature ice bath at a temperature of 10±2°C can slowly release organic solvents such as dichloromethane, thereby avoiding cracks or wrinkles on the surface of the microspheres, and stirring the primary emulsion can reduce collisions between emulsion droplets, thereby making it less likely to stick together.

[0126] The PLA microspheres prepared by the preparation method provided by the embodiment of the present invention have smooth and regular surfaces, no cracks, are not easy to stick together, have a narrow particle size distribution, and have a high yield of PLA microspheres.

[0127] It will be appreciated by those skilled in the art that the features described in the various embodiments and / or claims of the present invention may be combined and / or combined in various ways, even if such combinations and / or combinations are not explicitly described in the present invention. In particular, the features described in the various embodiments and / or claims of the present invention may be combined and / or combined in various ways without departing from the spirit and teachings of the present invention. All of these combinations and / or combinations fall within the scope of the present invention.

[0128] The embodiments of the present invention are described above. However, these embodiments are only for the purpose of illustration, and are not intended to limit the scope of the present invention. Although each embodiment is described above, it does not mean that the measures in each embodiment cannot be used in combination. The scope of the present invention is defined by the attached claims and their equivalents. Without departing from the scope of the present invention, those skilled in the art may make various substitutions and modifications, which should all fall within the scope of the present invention.

Claims

1. A method for preparing narrow distribution PLA microspheres, characterized in that: The preparation method comprises: Step (1): dissolving a PLA material in an organic solvent to obtain a dispersed phase; based on the total volume of the dispersed phase, the concentration of the PLA material in the dispersed phase is 60 to 300 mg / mL; Step (2): dissolving polyvinyl alcohol in hot water to obtain a continuous phase; based on the total weight of the hot water as 100%, the mass concentration of the polyvinyl alcohol in the continuous phase is 1.5-5%; Step (3): using a porous membrane tube to form a stable primary emulsion from the dispersed phase and the continuous phase through a membrane emulsification method; Step (4): stirring the primary emulsion in an ice bath at a temperature of 10±2° C. to remove the organic solvent and obtain a solidified emulsion; Step (5): the solidified emulsion is centrifuged, sieved and collected, and freeze-dried in sequence to obtain the narrow distribution PLA microspheres.

2. The preparation method according to claim 1, characterized in that: In step (1), the weight average molecular weight of the PLA material is 15 to 250 kDa.

3. The preparation method according to claim 1 or 2, characterized in that: In step (1), the PLA material includes one or a combination of L-polylactic acid, D-polylactic acid, racemic polylactic acid and meso polylactic acid.

4. The preparation method according to claim 1 or 2, characterized in that: In step (1), the organic solvent includes one or a combination of dichloromethane, chloroform, methyl formate, ethyl acetate, methyl acetate, acetone, dichloroethane and pentane; preferably dichloromethane.

5. The preparation method according to claim 1, characterized in that: In step (2), the hot water includes drinking water, purified water or water for injection, preferably purified water.

6. The preparation method according to claim 1 or 5, characterized in that: In step (2), based on the total weight of the continuous phase being 100%, the mass percentage of polyvinyl alcohol in the continuous phase is 1.5-3%.

7. The preparation method according to claim 1, characterized in that: In step (3), the volume ratio of the dispersed phase to the continuous phase is 1:2 to 200, preferably 1:

10.

8. The preparation method according to claim 1, characterized in that: In step (3), the pore size of the porous membrane tube is 0.1 to 20 μm, preferably 4 to 10 μm, and more preferably 7 μm.

9. The preparation method according to any one of claims 1, 7-8, characterized in that: In step (3), the porous membrane tube is a porous glass membrane tube.

10. The preparation method according to claim 1, characterized in that: Step (4) includes the following specific steps: The three-necked flask is fixed in a fume hood, an overhead stirrer is set up, a polytetrafluoroethylene stirring paddle is inserted into the stirrer, and the polytetrafluoroethylene stirring paddle is extended into the primary emulsion through the middle opening of the three-necked flask, and a side opening of the three-necked flask is connected to a circulating water vacuum pump; The primary emulsion was poured into the three-necked flask through the other side opening of the three-necked flask and the side opening was plugged with a glass piston. The primary emulsion was stirred in an ice bath at 10±2° C. to remove the organic solvent and obtain a solidified emulsion.

11. The preparation method according to claim 10, characterized in that: The polytetrafluoroethylene stirring paddle extends into the primary emulsion at a position of 1 / 4-3 / 4 of the height of the primary emulsion; And / or the stirring speed is 120-180 r / min, and the stirring time is 8-16 h.

12. A narrow distribution PLA microsphere, characterized in that: The narrow distribution PLA microspheres are prepared by the preparation method of narrow distribution PLA microspheres according to any one of claims 1 to 11, and have a particle size range of 10 to 30 μm, and a diameter distance of no more than 0.6, preferably no more than 0.

560.

13. Use of the narrow distribution PLA microspheres according to claim 12 as cosmetic plastic surgery fillers, as drug carriers for preparing sustained-release drugs, or as raw materials for preparing tissue engineering scaffolds.

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